JP2022507837A - Radiation protection nanoparticles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide radiation-protected nanoparticles. The radiation-protected nanoparticles of the present invention include first metal oxide nanoparticles. The radiation-protected nanoparticles further include a second metal oxide layer formed on the surface of the first metal oxide nanoparticles. The radiation-protected nanoparticles have a polar interface between the first metal oxide nanoparticles and the second metal oxide layer. [Selection diagram] Fig. 2

Description

The present invention relates to radiation protected nanoparticles.

Exposure of large volumes of ionizing radiation (IR) can occur in various medical fields and industries. In the medical field, more than 50% of cancer patients receive radiation therapy for cost-effectiveness and non-invasiveness. However, during radiation therapy, normal tissue can be damaged, especially if it is close to a tumor mass. One example is radiation-induced salivation or xerostomia, which occurs when salivary glands are exposed to radiation during radiation therapy for head and neck cancer (HNC).

Head and neck cancer is the eighth most common cancer, with 550,000 new cases reported each year, and 52% of head and neck cancer patients receive radiation therapy. Radiation-induced dry myositis is the largest acute and organ side effect in radiation therapy for head and neck cancer and can significantly affect the prognosis and long-term quality of life of patients. Chewing and swallowing, oral hygiene. It is related to major oral functions such as. In addition to the medical field, the use of nuclear energy in the industrial field is rapidly increasing as energy demand increases.

Unlike exposure to IR in the medical process, accidental total body irradiation (TBI) has serious life-threatening consequences such as hematopoietic and gastrointestinal syndromes. Microscopically, reactive oxygen species are rapidly produced within milliseconds via radiolysis of water when tissue is exposed to IR. Such reactive oxygen species react with nucleic acids and cell components to destroy biological functions and, on top of that, induce cell death. Because cells with high mitotic rates are more vulnerable to IR-induced reactive oxygen species, ancestral cells / stem cells or tissues involved in regeneration are usually damaged, and radiation damage is irreversible and acts to prevent regeneration. ..

In order to solve the above-mentioned problems, the present invention provides radiation-protected nanoparticles having an excellent radiation-protecting function.

The present invention provides radiation-protected nanoparticles with excellent biocompatibility.

Other objects of the invention will become clear from the following detailed description and accompanying drawings.

The radiation protected nanoparticles according to one embodiment of the present invention include the first metal oxide nanoparticles.

The radiation-protected nanoparticles further include a second metal oxide layer formed on the surface of the first metal oxide nanoparticles.

The radiation-protected nanoparticles have a polar interface between the first metal oxide nanoparticles and the second metal oxide layer. The polar interface comprises pairing of (the metal ion of the first metal oxide) -O- (the metal ion of the second metal oxide).

The second metal oxide layer is formed by epitaxially growing on the surface of the first metal oxide nanoparticles.

The second metal oxide layer increases oxygen voids on the surface of the first metal oxide nanoparticles.

The second metal oxide layer is strained to expose the {332} surface.

The first metal oxide contains ceria and the second metal oxide contains manganese oxide.

The first metal oxide nanoparticles have oxygen voids. The first metal oxide nanoparticles have nanocrystals having a fluorite structure.

The radiation-protected nanoparticles according to the examples of the present invention can have excellent radiation protection ability and biocompatibility. The radiation-protected nanoparticles can reduce systemic toxicity while increasing the ability to scavenge reactive oxygen species. In addition, the radiation-protected nanoparticles can have long-lasting stable activity and low active dose. The radiation-protected nanoparticles can be used locally and systemically to prevent various radiation-induced damage. Not only are the radiation-protected nanoparticles non-toxic or small, but even small amounts can directly protect tissues and cells from radiation and promote rapid regeneration and recovery of function.

The radiation protection nanoparticles according to one Example of this invention are shown. The radiation-protected nanoparticles according to another embodiment of the present invention are shown. It is a figure for demonstrating the characteristic of the radiation protection nanoparticles of FIGS. 1 and 2. It is a figure for demonstrating the characteristic of the radiation protection nanoparticles of FIGS. 1 and 2. It is a figure for demonstrating the characteristic of the radiation protection nanoparticles of FIGS. 1 and 2. It is a figure for demonstrating the characteristic of the radiation protection nanoparticles of FIGS. 1 and 2. It is a figure for demonstrating the characteristic of the radiation protection nanoparticles of FIGS. 1 and 2. It is a figure for demonstrating the characteristic of the radiation protection nanoparticles of FIGS. 1 and 2. It is a figure for demonstrating the characteristic of the radiation protection nanoparticles of FIGS. 1 and 2. It is a figure for demonstrating the characteristic of the radiation protection nanoparticles of FIGS. 1 and 2. It is a figure for demonstrating the enzyme-like activity of cerium-manganese oxide nanoparticles. It is a figure for demonstrating the enzyme-like activity of cerium-manganese oxide nanoparticles. It is a figure for demonstrating the enzyme-like activity of cerium-manganese oxide nanoparticles. It is a figure for demonstrating the enzyme-like activity of cerium-manganese oxide nanoparticles. It is a figure for demonstrating the enzyme-like activity of cerium-manganese oxide nanoparticles. It is a figure for demonstrating the enzyme-like activity of cerium-manganese oxide nanoparticles. It is a figure for demonstrating the enzyme-like activity of cerium-manganese oxide nanoparticles. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against eSMG from radiation-induced damage. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against eSMG from radiation-induced damage. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against eSMG from radiation-induced damage. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against eSMG from radiation-induced damage. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against eSMG from radiation-induced damage. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against eSMG from radiation-induced damage. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against eSMG from radiation-induced damage. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against IR which is totally irradiated in the living body.

Hereinafter, the present invention will be described in detail with reference to examples. The object, features and advantages of the present invention can be easily understood from the following examples. The present invention is not limited to the examples described herein, and may be embodied in other embodiments. In the examples presented herein, the ideas of the present invention are fully communicated so that the disclosed contents are thorough and complete, and to a person having ordinary knowledge in the technical field to which the present invention belongs. It is provided to ensure that. Therefore, the present invention should not be limited by the following examples.

In the present specification, terms such as first and second have been used to describe various elements, but the elements should not be limited by such terms. This term has only been used to distinguish the elements from each other. Also, when one element is mentioned to be on another, it means that it can be formed directly on another element, or a third element can intervene between them. do.

The terms + IR and IR + used in this specification and the accompanying drawings mean that they were exposed to radiation, -IR and IR- mean that they were not exposed to radiation, and + CM and CM + mean cerium-. It means that it has been treated with manganese oxide nanoparticles (eg, administered or injected into a living body), meaning that -CM and CM- have not been treated with cerium-manganese oxide nanoparticles. For example, + IR / + CM and IR + CM + indicate groups that have been exposed to radiation and treated with cerium-manganese oxide nanoparticles, and + IR / -CM and IR + CM- indicate groups that have been irradiated but treated with cerium-manganese oxide nanoparticles. Indicates a group that is not processed by.

The radiation protected nanoparticles according to one embodiment of the present invention include the first metal oxide nanoparticles. The first metal oxide nanoparticles have oxygen voids.

The first metal oxide nanoparticles have nanocrystals having a fluorite structure. The first metal oxide includes, for example, ceria (cerium oxide).

The radiation-protected nanoparticles according to another embodiment of the present invention include a first metal oxide nanoparticles and a second metal oxide layer formed on the surface of the first metal oxide nanoparticles.

The radiation-protected nanoparticles have a polar interface between the first metal oxide nanoparticles and the second metal oxide layer. The polar interface comprises pairing of (the metal ion of the first metal oxide) -O- (the metal ion of the second metal oxide). For example, the first metal oxide contains ceria, the second metal oxide contains manganese oxide, and the polar interface comprises Ce ⁴⁺ -O-Mn ²⁺ pairing. That is, the radiation-protected nanoparticles are ceria (CeO ₂ ) nanoparticles, a manganese oxide (Mn ₃ O ₄ ) layer formed on the surface of the ceria nanoparticles, and between the ceria nanoparticles and the manganese oxide layer. Includes Ce ⁴⁺ -O-Mn ²⁺ pairing at the hetero interface of.

The first metal oxide nanoparticles have oxygen voids.

The first metal oxide nanoparticles have nanocrystals having a fluorite structure. The second metal oxide layer is formed by epitaxially growing on the surface of the first metal oxide nanoparticles. The second metal oxide layer increases oxygen voids on the surface of the first metal oxide nanoparticles. The second metal oxide layer is strained to expose the {332} surface.

The radiation-protected nanoparticles have an F _{2 g} peak and an A _{1 g} peak in a visible light Raman spectrum. The surface oxygen reduction peak of the radiation-protected nanoparticles is higher than the temperature of the surface oxygen reduction peaks of the first metal oxide and the _second metal oxide in H2 TPR (temperature-projected reduction, TPR). Occurs at low temperatures.

FIG. 1 shows radiation-protected nanoparticles according to an embodiment of the present invention.

Referring to FIG. 1, the radiation protected nanoparticles include ceria nanoparticles. The ceria nanoparticles can have oxygen voids. The ceria nanoparticles have nanocrystals with a fluorite structure, and the lattice spacing of the (111) plane is 3.14 Å.

The ceria nanoparticles can be formed by the following method.

A mixed solution of 0.43 g of cerium nitrate (III), 2.7 g of oleylamine and 0.03 g of oleic acid in 15 mL of 1-dodecanol is heated to 120 ° C. in air. Aged for a few minutes until the mixed solution turns yellow. In the heating process, add 0.3 mL of distilled water at about 90 ° C. After the reaction, excess acetone, ethanol and acetonitrile are added to purify the ceria nanoparticles. The ceria nanoparticles are separated and collected by centrifugation. Thus, the microemulsion method can be used to form 4 nm sized, truncated octahedral ceria nanoparticles, predominantly surrounded by {100} and {111} planes (planes) in 1-dodecanol solution. can.

FIG. 2 shows radiation-protected nanoparticles according to another embodiment of the present invention.

Referring to FIG. 2, the radiation protected nanoparticles include cerium-manganese oxide nanoparticles. The cerium-manganese oxide nanoparticles include cerium oxide (ceria) and manganese oxide. The cerium-manganese oxide nanoparticles include ceria nanoparticles and a manganese oxide layer formed on the surface of the ceria nanoparticles.

The cerium-manganese oxide nanoparticles have a polar interface between the ceria nanoparticles and the manganese oxide layer. The polar interface comprises Ce ⁴⁺ -O-Mn ²⁺ pairing. That is, the cerium-manganese oxide nanoparticles are ceria (CeO ₂ ) nanoparticles, a manganese oxide (Mn ₃ O ₄ ) layer formed on the surface of the ceria nanoparticles, and the ceria nanoparticles and the manganese oxide layer. Ce ⁴⁺ -O-Mn ²⁺ pairing at the hetero interface between and can be included.

The ceria nanoparticles have oxygen voids. The ceria nanoparticles have nanocrystals with a fluorite structure. The manganese oxide layer can be formed by epitaxially growing on the surface of the ceria nanoparticles. The manganese oxide layer can increase the oxygen voids on the surface of the ceria nanoparticles. The manganese oxide layer can be strained to expose the {332} surface. The cerium-manganese oxide nanoparticles have a strain-induced high index Mn ₃ O ₄ {332} facets. The HAADF-STEM (High-angle annular dark-field scanning microscopic) image of FIG. 2 shows that the morphology of ceria nanoparticles is well preserved even after heterogeneous deposition of manganese oxide.

The cerium-manganese oxide nanoparticles can be formed by the following method.

A mixed solution of 0.09 g of ceria nanoparticles, 1.34 g of oleylamine, 0.14 g of oleic acid, 0.26 mL of hydrochloric acid, and 15 mL of xylene is heated to 90 ° C. A 0.8 mL aqueous solution of 0.38 M manganese chloride (II) is rapidly injected into the heated mixed solution. After aging the mixed solution for 2 hours, the cerium-manganese oxide nanoparticles are washed with hexane and ethanol, separated by centrifugation and collected. In this way, cerium-manganese oxide nanoparticles having a heterostructure can be formed by reacting the ceria nanoparticles with Mn ²⁺ ions. The heterostructured cerium-manganese oxide nanoparticles are well dispersed in chloroform.

The crystalline phase of ceria and heterostructured cerium-manganese oxide nanoparticles can be characterized by XRD (X-ray diffraction). The XRD pattern of the cerium-manganese oxide nanoparticles is almost the same as that of the ceria nanoparticles, which means that the manganese oxide was epitaxially grown on the ceria nanoparticles without uniform precipitation.

3 to 10 are diagrams for explaining the characteristics of the radiation-protected nanoparticles of FIGS. 1 and 2.

The manganese oxide (Mn ₃ O ₄ ) layer is the {112} plane of the triatomic layer when the thickness is about 1 nm and the molar ratio of Mn to Ce is 15-30%. The growth mechanism of manganese oxide on ceria can be analyzed through XPS (X-ray photoelectron spectroscopy), XAS (X-ray absorption spectroscopy) and STEM. Ceria (CeO ₂ ) nanoparticles with 45.8% Ce ³⁺ are slightly oxidized after reaction with Mn ²⁺ to have 41.3% Ce ³⁺ , as evidenced by XPS (FIG. 3).

When Mn of 15% or less as compared with Ce is deposited on the ceria nanoparticles, the oxidation state of Mn is almost Mn ²⁺ , forming a Ce ⁴⁺ -O-Mn ²⁺ polar interface (FIG. 4). However, in the case of manganese oxide with 1 to 3 atomic layers, both Mn ²⁺ and Mn ³⁺ are observed. Combining XPS, XAS data and known lattice parameters with the atomic arrangement of Mn ₃ O ₄ , the heterostructured cerium-manganese oxide nanoparticles (CemNNC) are CeO ₂ , Ce ⁴⁺ -O-Mn ²⁺ at the hetero interface. Pairing and Mn ₃ O ₄ . This is supported by the visible light (532 nm) Raman spectrum (FIG. 5). The spectra of cerium-manganese oxide nanoparticles show both the primary F _2g symmetry mode of CeO ₂ (452cm ^-1 ) and the A _1g symmetric stretching Mn-O binding peak of Mn ₃ O ₄ (645cm ^-1 ). .. The symmetry reduction of Ce—O and Mn—O bonds at the hetero interface between CeO ₂ and Mn ₃ O ₄ can explain the expansion of the peak. The asymmetric peak expansion and red shift of the F _2g peak indicates that the number of defects essential for removing reactive oxygen species is even higher.

The expanded A _{1 g} peak is due to the epitaxial strain of the Mn ₃ O ₄ layer induced by the polar interface (Ce ⁴⁺ -O-Mn ²⁺ ) and the lattice mismatch. The morphology of the Mn ₃ O ₄ layer can also support epitaxial strains. Since the lattice mismatch between the CeO ₂ (111) plane and the Mn ₃ O ₄ (112) plane is only about 1.2%, the Mn ₃ O ₄ is in the [112] direction parallel to the CeO ₂ (111) plane. Can grow into.

High exponential faceted Mn ₃ O ₄ {332}, which is a step on the {112} surface, can be exposed to minimize strain due to large lattice mismatches due to other surfaces (FIG. 2). The UV (325 nm) Raman spectrum is used to compare surface defects between CeO2 nanoparticles and cerium _- manganese oxide nanoparticles (FIG. 6). The intensity ratio of defect induction ( _D mode) peaks to F _2g peaks (ID / _IF 2g) at 598 cm ^-1 increased after deposition of Mn ₃ O ₄ , which increased oxygen voids on the surface of CeO ₂ . means.

Cerium-manganese oxide nanoparticles with abundant oxygen voids show high activity in scavenging active oxygen species, which is because cerium-manganese multi-metal oxide nanoparticles have a slightly lower concentration of Ce ³⁺ than CeO ₂ . Nevertheless, it can be due to charge delocalization for the entire nanoparticles (there is no Ce3 ⁺ site in chemically active colloidal ceria nanoparticles). The surface of the defective oxide can also be detected by the O1s XPS spectrum (Fig. 7).

The binding energies of about 529.2 eV, about 531.5 eV and 533.2 eV are allocated to lattice oxygen ( _Olatt ), surface oxygen ( _Oads ), and chemisorbed species ( _Osurf ) such as water, respectively. Can be Since the range of 531 to 532 eV indicates defective sites with electrophilic O ₂ ^2- , O ^2- and O ^- on the surface, the oxygen defect concentration can be estimated from the O _ads / O _latt ratio. can.

When the ratio of cerium-manganese oxide nanoparticles (2.16) was higher than CeO ₂ (1.04), the oxygen void level was higher in the cerium-manganese oxide nanoparticles, and the results obtained by Raman analysis. Match. H ₂ TPR (temperature-measured reduction, TPR) was measured to analyze the surface reducibility (FIG. 8). The surface oxygen reduction peak at 507 ° C for CeO ₂ and the Mn _{3O 4 reduction} peak for Mn ²⁺ at 498 ° C shift to a lower temperature (382 ° C), which is the surface reducing property for cerium-manganese oxide nanoparticles. Show improvement.

According to the results of TEM, XPS, XAS, Raman and H2 TPR, several layers of Mn ₃ O ₄ in addition to the Ce ⁴⁺ -O-Mn ₂ ⁺ polar interface formed by the heteroepitaxial step are cerium-manganese oxide nanoparticles. Required to form a high redox active surface of the particles.

9 and 10 show the catalytic activity of CeO ₂ , cerium-manganese oxide nanoparticles and Mn ₃ O ₄ on the reduction of H ₂ O ₂ and oxygen. 9 and 10 are on glassy carbon electrodes (GCE) in Ar saturated phosphate buffered saline (PBS, 0.01 M) in the presence of 5.00 mM H ₂ O ₂ and O ₂ saturated PBS, respectively. The circulating voltage current (CV) of the three supported samples is shown. Cerium-manganese oxide nanoparticles show a sharp increase in reduction current below −0.4 V and show improved catalytic activity compared to the single metals CeO ₂ and Mn ₃ O ₄ .

11 to 17 are diagrams for explaining the enzyme-like activity of cerium-manganese oxide nanoparticles.

Cerium-manganese oxide nanoparticles (Cemn) are converted to water-dispersible hydrophilic nanoparticles using a PEGylation method (FIG. 11). The PEGylated cerium-manganese oxide nanoparticles show a hydrodynamic diameter of 9-15 nm in PBS (Phosphate-Buffered Saline) (FIG. 12). Analyzing the cell viability based on the concentration of cerium-manganese oxide nanoparticles, cerium-manganese oxide nanoparticles have excellent biocompatibility (FIG. 13).

The enzymatic activity of ceria nanoparticles (CeO ₂ ) and cerium-manganese oxide nanoparticles (Cemn) in an aqueous medium is representative of reactive oxygen species using SOD, CAT mimicry and HORAC (hydroxyl radical antioxidant capacity) analysis methods. , O ^2- , H ₂ O ₂ , · OH (FIGS. 14 to 16).

Cerium-manganese oxide nanoparticles (Cemn) exhibit superior enzymatic performance in three representative reactive oxygen species compared to CeO ₂ or Mn ₃ O ₄ nanoparticles. This is because the Mn ions introduced into the CeO ₂ nanoparticles increase the oxygen voids on the surface of the CeO ₂ and the reducibility of the cerium-manganese oxide nanoparticles. Among them, the cerium-manganese oxide nanoparticles having a thick Mn layer can expose a site having a higher exponential facet Mn {332} than a thin Mn layer to provide a site having high catalytic activity, and thus have excellent enzymatic performance. Is shown.

In order to analyze the scavenging ability of reactive oxygen species of cerium-manganese oxide nanoparticles, intracellular levels of reactive oxygen species are measured using a fluorescent probe (FIG. 17). Strong fluorescence signals are observed in cells treated with tBHP alone, with significantly increased levels of reactive oxygen species compared to the control group. However, this fluorescence signal is greatly suppressed when tBHP is treated with CeO ₂ and further suppressed when treated with cerium-manganese oxide nanoparticles (CemNNC), and the CeO ₂ nanoparticles and cerium-manganese oxide nanoparticles. The particles exhibit antioxidant capacity, and the cerium _- manganese oxide nanoparticles exhibit superior scavenging capacity to the CeO2 nanoparticles at the cellular level.

18 to 24 are diagrams for explaining the protection of cerium-manganese oxide nanoparticles against eSMG from radiation-induced damage.

An organ in vitro culture model that has many advantages over other models to investigate the radioprotective capacity of the serum-free germ submandibular gland (eSMG) of cerium-manganese oxide nanoparticles (Cemn). It was used. For example, the in vitro model cannot reflect the interactions between many cell types, and the in vivo salivary gland IR injury model consumes a lot of time and resources. However, eSMG models include progenitor cells, Vascural endothelial (VE) cells, epithelial acinar / ductal cells, mesenchymal cells, and accessory cells. It only takes 48-72 hours to investigate IR-induced damage while maintaining complex biological interactions between various cell types, including parasypthetic ganglion cells) (FIGS. 18 and 19).

The intracellular reactive oxygen species level was measured through CellRox Green, and the reactive oxygen species level in the epithelial bud was significantly lower in the cerium-manganese oxide nanoparticles-administered group than in the control group. This indicates that the cerium-manganese oxide nanoparticles effectively remove the reactive oxygen species produced by IR (FIG. 20). C-kit + salivary gland progenitor cells survive by interacting with niche and surrounding microenvironments such as mesenchymal cells, parasympathetic ganglia (PSG), vascular endothelium (VE) cells. And maintain self-renewal ability.

When salivary gland asiner cells are damaged, c-kit + ancestral cells are reactivated from dormant state and differentiated into new asiner cells. Therefore, protecting ancestral cells and their niche from radiation damage is important for salivary gland regeneration.

Therefore, it is possible to confirm the radioprotective effect of cerium-manganese oxide nanoparticles on epithelial bacterial tube cells, VE cells, PSG and c-kit + ancestral cells through immunohistological analysis. The asiner / tube cell ratio was not maintained constant in the irradiated human salivary glands. This phenomenon was also observed in the mouse eSMG model. The eSMG irradiated with 13 Gy had a significantly reduced asiner (AQP5 +) / tube (K19 +) cell ratio, while the cerium-manganese oxide nanoparticles treated group showed an improved asiner / tube cell balance.

PSG growth and branching were significantly suppressed in the + IR / -CM group, but the + IR / + CM group restored PSG growth to normal levels. The rate of apoptosis of total explants was measured by caspase 3/7 Glo analysis. The + IR / -CM group showed a significant increase in caspase 3/7 activity over + IR / + CM, which effectively protects IR-induced apoptosis of cerium-manganese oxide nanoparticles. Show to do.

In each cell type, active caspase 3-positive cells were counted to measure the cell killing rate of asiner cells, PSG and c-kit + progenitor cells, and in all three types of cells, the + IR / + CM group was + IR /-. The results showed that the cell death rate was significantly lower than that of the CM group. In the + IR / -CM group, the number of c-kit + ancestral cells was significantly reduced, but the c-kit + cells were well conserved in the + IR / + CM group.

The c-kit + ancestral cells that survived in the + IR / + CM group were later successfully differentiated into AQP5 + Acinar cells, whereas the + IR / -CM group significantly reduced the number of differentiated Acinar cells (FIG. 21). ..

The protective efficacy of cerium-manganese oxide nanoparticles was quantitatively confirmed by the mRNA expression level against acinar cells (AQP5, CHRM1, CHRM3), PSG (TUJ1, NRTN) and c-kit + ancestral cells (C-kit) ( 22 to 24).

The expression levels of all markers in the CeMn-treated eSMG group were higher than those in the untreated group, which is similar to immunofluorescence data. These results provide cerium-manganese oxide nanoparticles with overall radiation protection for progenitor cells and their surrounding structures to help progenitor cells survive and proliferate and differentiate into functional asiner cells. Is shown.

To investigate the overall vicious circle of biological events that occur after IR, the overall gene expression levels of each group (mRNA extracted from 10 eSMGs per group) are compared via next-generation sequencing. did. Increased reactive oxygen species by IR can induce immune response, inflammation and cell death. Cell debris of dead cells increases inflammation, and reactive oxygen species levels can induce more cell death. IR induced a rapid increase in various genes for reactive oxygen species, inflammatory response, immune response and cell killing, but treatment with cerium-manganese oxide nanoparticles mitigated the expression level of such genes. .. The eSMG organism model lacks circulating immune cells, but partially reflects IR-induced acute inflammation and immune responses, and biological processes for radiation-induced vicious circles have also been successful with cerium-manganese oxide nanoparticles. Was blocked.

25 to 35 are diagrams for explaining the protection of cerium-manganese oxide nanoparticles against IR locally irradiated in the living body, and FIGS. 36 to 49 show the whole living body being irradiated. It is a figure for demonstrating the protection of the cerium-manganese oxide nanoparticles against the IR.

The ability of cerium-manganese oxide nanoparticles (Cemn) to protect the structure and function of SMGs in the body and oral and general health against IR-induced xerostomia were tested. Cerium-manganese oxide nanoparticles were injected into two SMGs of ICR mice via each tube prior to IR. The position of the SMG was visually confirmed, and X-ray irradiation was performed in an amount of 5 Gy per day for 4 days (FIG. 25).

After 90 days, the mice underwent salivary gland function testing and histological analysis. The salivary gland function test was performed by collecting whole saliva from mice stimulated with pilocarpine. The average saliva flow velocity after IR decreased from 3.5 μl / min to 1.2 μl / min, but the saliva flow velocity increased significantly to 2.5 μl / min in the cerium-manganese oxide nanoparticles administration group (Fig.). 26).

Since it is important that saliva completely envelops oral tissues such as the tongue and buccal mucosa, the area of the oral cavity covered by the saliva produced was measured with sodium fluorescein paper (Fig. 27). Little fluorescent dye was observed in the oral cavity of the + IR / -CM group, whereas in the + IR / + CM group, fluorescent dye was found in the abdomen and back of the tongue, palate, and buccal mucosa.

The amount of fluorescein dye remaining on the removed paper was confirmed with a spectrophotometer. About 20% of the dye was washed away by saliva in the -IR / -CM and + IR / + CM groups, but almost no dye was diffused in the + IR / -CM group (FIG. 28).

The histopathological changes in SMG induced by IR were investigated (Fig. 29). A significant increase in leukocyte infiltration and tissue fibrosis was observed in the + IR / -CM group. However, the + IR / + CM group showed no significant difference in leukocyte infiltration or fibrous changes compared to the -IR / -CM group.

Secretory acinar cells were visualized by PAS staining (FIG. 30). Almost no asiner cells were observed in the + IR / -CM group, but the number of asiner cells increased significantly in + IR / + CM. The area of acinar / tube cells was quantified and the + IR / -CM group showed a significantly reduced acinar / ductal ratio as observed in the eSMG IR model.

However, the damaged asinar / tube cell ratio was partially restored in the + IR / + CM group. Actual cell death of SMG tissue was examined by TUNEL assay. As presented, the TUNEL + cell count was significantly reduced in the CeMn-treated group (FIGS. 31 and 32). Radiation-induced xerostomia causes inflammation of the oral tissue, loss of taste, destruction of microbes in the oral cavity, and difficulty swallowing, making it difficult for patients to eat food.

As a result of the conversion from local IR to SMG, body weight was significantly reduced 4 weeks after IR. This is similar to clinical symptoms. However, mice treated with cerium-manganese oxide nanoparticles did not show significant weight loss (FIG. 33). The tongue of IR mice showed dull thread papillae-like protrusions and a thick keratinized layer. However, the tongues of the cerium-manganese oxide nanoparticle treatment group showed the coexistence of normal-thick keratinized layers with normal and blunt filamentous papillae-like projections (FIG. 34).

The antibacterial activity of saliva is one of the most important functions in saliva. Oral bacterial samples were collected on a buccal swap and cultured for colonization analysis. An abnormally increased number of bacterial colonies was observed in the + IR / -CM group, but was partially significantly reduced in the + IR / + CM group (FIG. 35). These results indicate that locally injected cerium-manganese oxide nanoparticles can protect the structure and function of salivary glands and prevent various oral and systemic complications induced by salivary gland IR. ..

Mice were injected intraperitoneally with PBS or 50 mg / kg cerium-manganese oxide nanoparticles and body weight was measured at 3-day intervals for 30 days. The cerium-manganese oxide nanoparticles infused group did not lose significantly weight compared to the PBS infused group. Thirty days later, the mice were sacrificed and the organs were harvested for histopathological examination. In addition, no significant pathological changes were observed in mice treated with cerium-manganese oxide nanoparticles in seven major organs, including the viscera, kidneys, spleen, liver, heart, lungs, and bladder. This indicates that the cerium-manganese oxide nanoparticles do not show significant systemic toxicity. Next, the systemic protection ability of the cerium-manganese oxide nanoparticles was tested at a concentration of 0.55 mg / kg, which is 1/100 of the concentration of the cerium-manganese oxide nanoparticles used in the toxicity test.

Mice received 13 Gy total body irradiation (TBI) after injection of cerium-manganese oxide nanoparticles (FIG. 36). No mice died in the -IR / -CM and -IR / + CM groups. This indicates that the active capacity of the cerium-manganese oxide nanoparticles does not cause systemic toxicity. In the + IR / -CM group, the survival rate of mice decreased sharply to 50% on the 12th day after TBI, and all 15 mice died on the 15th day after TBI. However, 10 mice survived in the + IR / + CM group 150 days after TBI (Fig. 37).

To understand the factors that contributed to the increased viability of the treatment group of cerium-manganese oxide nanoparticles, blood and bone marrow cells (BMC), which are organs damaged mainly by acute radiation syndrome (ARS). ) And short-term and long-term pathophysiological changes in the small intestine were observed. It is known that the most serious response to radiation exposure is an increase in cytokines in the circulatory system that can induce nausea and fatigue.

Six hours after TBI, interleukin-1 beta (IL-1b), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α ( The serum levels of four blood cytokines, including TNF-a), were increased (FIG. 42). However, the treated group of cerium-manganese oxide nanoparticles had significantly less increase in four cytokines than the untreated group of cerium-manganese oxide nanoparticles. Mouse BMC cell killing rates were measured after TBI and BMC cell killing were significantly suppressed in the cerium-manganese oxide nanoparticles treatment group (FIGS. 38 and 39).

The total number of BMCs 1 day after TBI was also separated and factored (Fig. 40). Approximately 95% of BMC was removed in the untreated group of cerium-manganese oxide nanoparticles, whereas only an average of 50% of BMC was removed in the treated group of cerium-manganese oxide nanoparticles. Plasma MDA counts 1 day after TBI were measured. The treated group of cerium-manganese oxide nanoparticles significantly reduced the plasma MDA concentration compared to the untreated group of cerium-manganese oxide nanoparticles (FIG. 41).

On the 4th day after TBI, MDA values of 5 organs such as small intestine, liver, kidney, spleen, and lung were measured (FIG. 43). All other organs, except the liver, had significantly increased post-TBI MDA levels. No significant increase in MDA levels was observed in the small intestine, spleen and lungs of the cerium-manganese oxide nanoparticles treatment group compared to the -IR / -CM group.

Radiation-induced small intestinal injuries that could induce radiation damage were investigated (Fig. 44). One week after TBI, the duodenum was harvested and histopathologically examined. The villi were severely damaged by TBI, but the treated group of cerium-manganese oxide nanoparticles showed a relatively undamaged villous structure, and the villi length was even longer than the untreated group of cerium-manganese oxide nanoparticles. Was maintained (FIGS. 46 and 47).

Tanel assay and Ki-67 staining results also showed that cerium-manganese oxide nanoparticles could protect villi and crypto cells from IR and maintain mitotic activity of stem and progenitor cells in crypto cells (FIG. 45). ).

To confirm the self-renewal ability of surviving script cells to a treatment group of cerium-manganese oxide nanoparticles, the scrotum was isolated from mice within 30 minutes after TBI and cultured under in vitro organoid culture conditions. (Fig. 48). Gallbladder separated in the untreated group of cerium-manganese oxide nanoparticles after 7 days was unable to form intestinal organs, whereas the treated group of cerium-manganese oxide nanoparticles had significantly increased organ formation. The efficiency was shown (Fig. 49). In addition, the intestinal organs of the treatment group of cerium-manganese oxide nanoparticles exhibited features of the normal organoid phenotype, including central lumen, cryptostructure and villi. However, the scrotum isolated in the untreated group of cerium-manganese oxide nanoparticles was unable to form spherical cysts for 7 days.

Finally, organs were harvested from mice treated with cerium-manganese oxide nanoparticles that survived 150 days after TBI and evaluated for long-term damage. Both the duodenum and jejunum of surviving mice showed no signs of tumorigenesis or etiology.

The specific examples of the present invention have been considered above. Those who have ordinary knowledge in the technical field to which the present invention belongs will understand that the present invention can be embodied in a modified form within a range that does not deviate from the essential characteristics of the present invention. Therefore, the disclosed examples should be considered from a descriptive point of view, not from a limiting point of view. The scope of the invention is set forth in the claims, not the description described above, and all differences within the equivalent scope should be construed as included in the invention.

The radiation-protected nanoparticles according to the examples of the present invention can have excellent radiation protection ability and biocompatibility. The radiation-protected nanoparticles can reduce systemic toxicity while increasing the ability to scavenge reactive oxygen species. In addition, the radiation-protected nanoparticles can have long-lasting stable activity and low active dose. The radiation-protected nanoparticles can be used locally and systemically to prevent various radiation-induced damage. Not only are the radiation-protected nanoparticles non-toxic or small, but even small amounts can directly protect tissues and cells from radiation and promote rapid regeneration and recovery of function.

Claims (10)

Radiation-protected nanoparticles containing the first metal oxide nanoparticles. The radiation-protected nanoparticles according to claim 1, further comprising a second metal oxide layer formed on the surface of the first metal oxide nanoparticles. The radiation-protected nanoparticles according to claim 2, wherein the radiation-protected nanoparticles have a polar interface between the first metal oxide nanoparticles and the second metal oxide layer. The radiation according to claim 3, wherein the polar interface comprises pairing of (metal ion of the first metal oxide) -O- (metal ion of the second metal oxide). Protective nanoparticles. The radiation-protected nanoparticles according to claim 2, wherein the second metal oxide layer is formed by epitaxially growing on the surface of the first metal oxide nanoparticles. The radiation-protected nanoparticles according to claim 2, wherein the second metal oxide layer increases oxygen voids on the surface of the first metal oxide nanoparticles. The radiation-protected nanoparticles according to claim 2, wherein the second metal oxide layer is strained to expose a {332} surface. The radiation-protected nanoparticles according to claim 2, wherein the first metal oxide contains ceria and the second metal oxide contains a manganese oxide. The radiation-protected nanoparticles according to claim 1, wherein the first metal oxide nanoparticles have oxygen voids. The radiation-protected nanoparticles according to claim 1, wherein the first metal oxide nanoparticles have nanocrystals having a fluorite structure.

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